Shin KS , Rothberg BS , Yellen G .

HL57383 NHLBI NIH HHS

	Figure 1. Hyperpolarization-activated mHCN1 currents. (A) Representative recordings from inside-out patches excised from HEK293 cells expressing mHCN1. Currents were elicited by an 800-ms hyperpolarization from a holding voltage of +10 mV to voltages ranging from −140 to −60 mV in 10-mV increments. Voltage was returned to +30 mV for 150 ms to measure the tail currents. (B) Voltage dependence of channel activation. Initial tail current amplitudes at +30 mV (as shown in A) were normalized to the maximal tail current. Solid line shows the best fit of the data to a Boltzmann function; normalized current = 1/[1 + exp ((V1/2 − V)/slope)], where V is voltage in millivolts and V1/2 is the activation midpoint voltage. A mean V1/2 of −101.3 mV and a slope of e-fold change for 7.1 mV were determined from five experiments.
	Figure 2. Concentration dependence of ZD7288 blockade of mHCN1 currents. (A) Superimposed current records at −110 mV with different ZD7288 concentrations. ZD7288 was applied for 4 s, as indicated by the shaded bar. (B) Fractional blockade of mHCN1 at −110 mV versus ZD7288 concentration. The solid line is the best fit with the equation f = [ZD]/([ZD] + K d). The fitted K d was 40.5 ± 3.3 μM (data points with n = 3–5).
	Figure 3. Voltage-dependent and open channel block of mHCN1 current. (A) Superimposed current record from −140 to −90 mV with 100 μM ZD7288. ZD7288 was applied for 4 s, as indicated by the shaded bar. (B) Fractional block with 100 μM ZD7288 measured at different voltages. The data were fitted with a Boltzmann equation with a V1/2 of −117.5 mV and zδ of 4.2 (n = 3–5). The dotted line shows the G-V of mHCN1 for comparison. (C) Open channel block of mHCN1 by ZD7288. ZD7288 (100 μM) was applied for 4 s at −70 or −110 mV during the period indicated by the bar, and the voltage was returned to +10 mV for 1 s to close the channels. Finally, a second activating pulse at −120 mV was applied to test the remaining blockade of the channel. For the control, the voltage step was applied without blocker present.
	Figure 4. Hyperpolarization-activated SPIH currents. (A) Recordings of SPIH currents in the presence or absence of cAMP. Currents were measured with excised inside-out patches from SPIH-expressing HEK293 cells in the presence or absence of cAMP (100 μM). Currents were elicited by a series of 800-ms hyperpolarizing voltage steps between −120 and −30 mV in 10-mV steps from a holding voltage of +10 mV. (B) Voltage dependence of channel activation. Tail current amplitudes at +30 mV in the presence of 100 μM cAMP (as shown in A) were normalized to the maximal tail current. The solid line shows the Boltzmann fit to the data with a mean midpoint voltage V1/2 of −73.5 mV and a slope of e-fold change for 5.4 mV (each point is the mean of five experiments).
	Figure 5. ZD7288 block of SPIH. (A) ZD7288 (100 μM) was applied to an excised inside-out patch for 1 s at −110 mV. Much of the current did not recover after removal of the blocker. (B)100 μM ZD7288 was repeatedly applied for 1 s at −110 mV at each time point (arrows). The inset shows the superimposed traces of each application. The traces are flipped over to show the reduction in the size of current because of the irreversible blockade by ZD7288. Dots indicate the steady-state current in response to an activating voltage step to −120 mV applied every 4 s between blocker applications. (C) Fractional remaining currents after application of ZD7288 (100 μM) were measured (as shown in B) and plotted against cumulative treatment time. The rate constant for the irreversible block was calculated from a time constant of a monoexponential fit to the data. In the same way, the rates at different voltages were determined. (D) Rate of irreversible block in SPIH. The rates were measured at different voltages (as described in B and C) in the presence of cAMP (square). The triangle indicates the rate of irreversible block at −110 mV in the absence of cAMP. For comparison, the normalized G-V relationship of SPIH is shown as open circles. All individual points give the mean and SEM of at least three determinations.
	Figure 7. Sequence alignment of the S5, P, and S6 regions. Amino acids that are identical between SPIH and mHCN1 are shown in bold. The three residues in the S6 region that are different from each other and appear to be critical in the reversibility of ZD7288 blockade are indicated by asterisks.
	Figure 6. Reversibility of ZD7288 blockade in different chimeras. 100 μM ZD7288 was repeatedly applied at −110 mV at each time point indicated by the bars (4 s for mHCN1, H-S/P, and mHCN1-χ3; 2 s for H-S/S5; and 1 s for SPIH and SPIH-χ3). Between the applications, 800 ms-long activating voltage steps to −140 mV were applied every 4 s, and the steady-state currents were measured. Dots indicate normalized currents to the maximal steady-state current level. The maximal currents at −140 mV were as follows: −30.2 pA for mHCN1; −526 pA for H-S/P; −650 pA for H-S/S5; −98.2 pA for mHCN1-χ3; −85 pA for SPIH; and −78.2 pA for SPIH-χ3. The current traces were also normalized to the maximal current level measured just before the first blocker application. Schematic representations of chimeras are shown to the left of each graph.
	Figure 8. Trapping of ZD7288 in SPIH-χ3 mutant. (Top) 100 μM ZD7288 was applied to the excised inside-out patch at −110 mV for 1 s to achieve maximal blockade, and the blocker was removed to check the kinetics of blocker unbinding. The dashed line is a monoexponential fit to the slowly recovering current after removal of blocker (time constant = 3.1 s). (Bottom) 100 μM ZD7288 was applied to the same patch as shown above at −110 mV for 1 s to achieve complete block. The blocker was removed and, at the same time, the voltage was returned to +10 mV to close the channels. After 5 s, the voltage was stepped back to −110 mV to check recovery from the closed blocked state. The time constant for this recovery (3.2 s) was very similar to the value obtained above.
	Scheme S1.
	Figure 9. Voltage dependence of the ZD7288 block predicted by two possible models. A fraction of the current not blocked at different voltages was measured at three different concentrations of ZD7288 (30, 100, and 500 μM). The solid lines are the fits with the preferential closed state block model (Fig. 2), where kg = exp[−(V − 111.8)/4.9], k−g= exp[−(V − 129.6) / 4.3], and K d = 1257.6 μM. The dashed lines show the fit using the two open state model (Fig. 2), where kg = exp[−(V − 95.6)/15.0], kt = exp[−(V − 97.5)/6.0], K d1 = 6.0 μM and K d2 = 1799.7 μM.
	Scheme S2.

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